6
to moderate yields and ee for 3a–d. Starting with another
than other enzymatic ones for the synthesis of vicinal diols
easily available class of substrates, racemic epoxides, EH
might be able to perform the enantioselective hydrolysis giving
the corresponding diol. However, no such bioconversions have
been reported for the preparation of (S)-3a–d in high ee, due to
the lack of the required extremely high enantioselectivity of
EH. Another enzymatic method for the preparation of vicinal
diols such as (S)-3a and (S)-3c in high ee involved the reduc-
(S)-3a–d in high ee. Further improvement of the productivity
involves the engineering and use of a highly active recombi-
nant biocatalyst expressing the epoxide hydrolase. The con-
cept demonstrated here is generally applicable to the
enantioselective dihydroxylation of other olefins by selecting
and combining the appropriate enzymes in a similar way. The
principles and knowledge obtained could also be useful for the
development of other tandem biocatalysts systems for other
types of transformations.
1
0
tion of the corresponding hydroxy ketone with a reductase.
However, hydroxy ketones are quite expensive and are usually
prepared by chemical oxidation of the corresponding diols.
The use of a tandem biocatalysts system also allows the
circumvention of negative effects that would otherwise arise
from the accumulation of an intermediary product—such as
styrene oxide in this case. By removing it quickly from the
aqueous phase through the subsequent hydrolysis, any inhibiting
effects—either by toxicity or enzyme inhibition—are reduced.
As shown in Fig. 3, the addition of EH to the reaction system
containing SMO accelerated the conversion of styrene 1a
Financial support by the Ministry of Education of Singapore
through an AcRF Tier 1 Grant (Project No.: R-279-000-239-112)
is gratefully acknowledged.
Notes and references
z Procedure for asymmetric dihydroxylation of styrene 1a with
tandem biocatalysts in one pot: cells of E. coli JM101 pSPZ10 and
Sphingomonas sp. HXN-200 were prepared according to published
8
a,9b
procedures.
Lyophilized CFE of Sphingomonas sp. HXN-200 was
ꢀ1
prepared by passing the cell suspension (25 g cdw L ) in KP buffer
(pH 7.5) through a homogenizer at 30 KPSi, removing the cell debris
by centrifuging at 245 000 g and 4 1C for 30 min, and lyophilizing
the CFE for 48 h. Lyophilized CFE (200 mg) was added to a
(
to the epoxide or to the diol, depending on the biocatalyst
composition) by a factor of 2 when compared to the experi-
ment without EH. Similar phenomena were observed for the
cases using chlorostyrenes 1b–d as substrates, even though the
reaction conditions had to be adapted due to the lower specific
activities of the catalysts for these substrates. These represent
one more advantage of using tandem biocatalysts in one
pot over the two separate catalysts, in addition to the lack
of need for separation and purification of the intermediate
products.
1
(
0 mL suspension of frozen–thawed cells of E. coli JM101 pSPZ10
2.5 g cdw L ) in 100 mM KP buffer (pH 7.5) containing glucose
ꢀ1
(28 mM), followed by the addition of 10 mL n-hexadecane containing
styrene 1a (20 mM). The mixture was incubated at 200 rpm and 30 1C.
The reaction was followed by analysing samples taken at different time
points (see ESIw). The ee of (S)-3a was determined by HPLC analysis
with a Chiralcel OB-H (250 mm ꢂ 4.6 mm) column: UV detection
at 210 nm; eluent: n-hexane and 2-propanol (95 : 5); flow rate:
ꢀ1
1
(
.0 mL min ; retention times: 14.7 and 18.7 min for (R)- and
S)-3a, respectively. After 35 h reaction, (S)-3a was formed in 95%
yield with 99.4% ee.
In summary, a tandem biocatalysts system was successfully
developed for the enantioselective dihydroxylation of aryl
olefins 1a–d, giving the corresponding diols 3a–d in good yield
and excellent ee. By utilizing the tandem biocatalysts in a two-
liquid phase system, higher conversion for the epoxidation was
achieved than with the use of only styrene monooxygenase; no
isolation of the epoxide intermediate is necessary, and the diol
product was easily separated. The dihydroxylation of aryl
olefins with tandem biocatalysts provides a better method
1
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Fig. 3 Conversion of 1a–d in the epoxidation of olefins by resting
cells of E. coli JM101 (pSPZ10) without (&)/with (’) lyophilized cell
free extract (LCFE) of Sphingomonas sp. HXN-200. 1a–d: 10 mM; 1a:
07; (b) Z. Y. Liu, J. Michel, Z. S. Wang, B. Witholt and Z. Li,
ꢀ ꢀ1 ꢀ1
1
ꢀ1
ꢀ1
2
.5 g L E. coli, 20 g L LCFE, 1 h; 1b: 5 g L E. coli, 35 g L
ꢀ1
ꢀ1
LCFE, 12 h; 1c: 5 g L E. coli, 40 g L LCFE, 12 h; 1d: 5 g L
10 T. Tsujigami, T. Sugai and H. Ohta, Tetrahedron: Asymmetry,
2001, 12, 2543.
ꢀ1
E. coli, 45 g L LCFE, 20 h.
This journal is ꢁc The Royal Society of Chemistry 2009
Chem. Commun., 2009, 1481–1483 | 1483